G protein-coupled receptors (GPCRs) mediate hormonal control of numerous signaling pathways, many of which are dynamically regulated. At the receptor level, regulation can occur via inhibition of GPCR/G protein coupling (desensitization), redistribution of cell surface receptors (trafficking), or receptor degradation (down-regulation). Two protein families, G protein-coupled receptor kinases (GRKs) and arrestins, play a critical role in these processes. GRKs specifically phosphorylate the activated GPCRs, which in turn pro- motes arrestin binding. Arrestin interaction has been directly linked to many regulatory processes including GPCR desensitization, trafficking, and signaling via non-receptor tyrosine kinase and MAP kinase pathways. In the previous grant period we focused on characterizing the interaction of arrestins with GPCRs; the role of arrestins in regulating GPCR trafficking; and the interaction of arrestins with additional proteins. During the course of this work we also initiated studies in C. elegans in an effort to better correlate the structure and function of arrestin with in vivo biology. In the present application, we propose to extend our efforts in two major directions. The first involves further defining the role of GPCRs in regulating arrestin function by characterizing a number of arrestin/protein interactions that we have recently identified. The second involves our continued use of C. elegans as a model system to better define the mechanism of arrestin function in vivo. These studies are aimed at addressing several important questions. Do GPCRs regulate interactions that contribute to the diverse nature of arrestin function? What interactions mediate arrestin function in vivo and how are such interactions regulated? Overall, our efforts will provide unique mechanistic insight into the biochemical, cellular and molecular function of arrestins and should prove useful in understanding diseases where GPCR signaling defects are observed. G protein-coupled receptors have been implicated in many diseases including cancer, HIV, hypertension, sensory defects, and various neuronal disorders, and are the target for -50% of the drugs currently on the market. This research is aimed at understanding arrestins, a family of proteins that play an essential role in controlling GPCR function.